title: Overexpression of hypoxia-inducible factor-1α exacerbates endothelial barrier dysfunction induced by hypoxia.
abstract: OBJECTIVE
  The mechanisms involved in endothelial barrier dysfunction induced by hypoxia are incompletely understood. There is debate about the role of hypoxia-inducible factor-1α (HIF-1α) in endothelial barrier disruption. The aim of this study was to investigate the effect of genetic overexpression of HIF-1α on barrier function and the underlying mechanisms in hypoxic endothelial cells.
  
  
  METHODS
  The plasmid pcDNA3.1/V5-His-HIF-1α was stably transfected into human endothelial cells. The cells were exposed to normoxia or hypoxia. The mRNA and protein expressions of HIF-1α were detected by RT-PCR and Western blot respectively. The barrier function was assessed by measuring the transendothelial electrical resistance (TER). The Western blot analysis was used to determine the protein expression of glucose transporter-1 (GLUT-1), zonular occludens-1 (ZO-1), occludin, and myosin light chain kinase (MLCK) in endothelial cells. The mRNA expression of proinflammatory cytokines was detected by qRT-PCR.
  
  
  RESULTS
  Genetic overexpression of HIF-1α significantly increased the mRNA and protein expression of HIF-1α in endothelial cells. The overexpression of HIF-1α enhanced the hypoxia-induced increase of HIF-1α and GLUT-1 protein expression. HIF-1α overexpression not only exacerbated hypoxia-induced endothelial barrier dysfunction but also augmented hypoxia-induced up-regulation of MLCK protein expression. HIF-1α overexpression also enhanced IL-1β, IL-6 and TNF-α mRNA expression.
  
  
  CONCLUSIONS
  We provide evidence that genetic overexpression of HIF-1α aggravates the hypoxia-induced endothelial barrier dysfunction via enhancing the up-regulation of MLCK protein expression caused by hypoxia, suggesting a potential role for HIF-1α in the pathogenesis of endothelial barrier dysfunction in hypoxia.
authors.affiliation: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University Chongqing, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Wang
authors.forename: Huabing
authors.initials: H
authors.lastname: Qi
authors.forename: Chunhong
authors.initials: C
authors.lastname: Sun
authors.forename: Wen
authors.initials: W
authors.lastname: He
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Fengjun
authors.initials: F
authors.lastname: Wang
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000600:Amino Acids, Dicarboxylic; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009219:Myosin-Light-Chain Kinase; C040947:oxalylglycine